The life cycle of retroviruses is characterized by two steps, which are carried out by two virally encoded enzymes, reverse transcriptase (RT) and integrase (IN). In the first of these steps, RT converts the single-stranded viral RNAs found in virions into a linear double-stranded DNA that is longer than the RNAs from which it is derived. In the second step, integrase (IN) inserts this linear viral DNA into the host genome. Both steps are essential for the retroviral life cycle; both RT and IN are key anti-HIV drug targets. The development of new broadly effective, low-toxicity anti-HIV drugs against such targets is one of the high-priority research goals of the NIH, in part because of problems with emerging drug resistance in the developing world. ____The conversion of retroviral genomic RNA into DNA involves the two enzymatic activities of RT: a polymerase that can copy either RNA or DNA, and a ribonuclease H (RNase H) that cleaves RNA if it is part of an RNA/DNA hybrid. Although the RNase H of RT is essential for viral replication, there are no anti-HIV drugs that target RNase H. The two clinically important classes of anti-RT drugs -- nucleoside analogs (NRTIs) and nonnucleoside RT inhibitors (NNRTIs) -- instead target the polymerase. ____A major focus of our work has been on the mechanism(s) of RT inhibitor resistance to these two classes of drugs. There is, at this point, a reasonably good understanding of the mechanism(s) of NNRTI resistance, and considerable progress has been made in understanding NRTI resistance, although some important issues remain. The NRTIs that are currently used to treat HIV 1 infections lack the 3'-OH found on normal deoxynucleosides. If an NRTI is incorporated into viral DNA by RT, polymerization is blocked. Because NRTIs can also be incorporated into the mitochondrial and nuclear DNA of host cells, these drugs can be toxic to patients, particularly because HIV drug therapy is usually lifelong. In contrast to NRTIs, NNRTIs are, as a group, relatively nontoxic, but are prone to the development of resistance. We have focused on understanding NRTI resistance and on developing NNRTIs that are more broadly effective against the known drug-resistant mutants of HIV-1. We have also recently turned our attention to the RNase H activity of HIV-1 RT, which could be an effective (and important) target for the development of new inhibitors and drugs. ____Two mutations, G112D and M230I, were selected in HIV-1 RT by a novel NNRTI that we developed (Compound 13). G112D is located near the HIV-1 polymerase active site; M230I is located near the hydrophobic region where NNRTIs bind. Thus, M230I could directly interfere with NNRTI binding but G112D could not. Biochemical and virological assays were performed to analyze the effects of these mutations individually and in combination. M230I alone caused a reduction in susceptibility to NNRTIs while G112D alone did not. The G112D/M230I double mutant was less susceptible to NNRTIs than was M230I alone. In contrast, both mutations affected the ability of RT to incorporate nucleoside analogs. We suggest that the mutations interact with each other via the bound nucleic acid substrate; the nucleic acid forms part of the polymerase active site, which is near G112D. The positioning of the nucleic acid is influenced by its interactions with the primer grip region and could be influenced by the M230I mutation. We suggest that the unusual mutation in HIV-1 RT selected by Compound 13 in culture is a good sign in terms of its possible clinical utility. Although the increased use of antiretroviral therapy globally is a very promising development, it carries with it an increase in the worldwide burden of drug-resistant HIV-1 mutants. NNRTIs in general, and RPV in particular, are an important component of the drug combinations that are in general use. Having new NNRTIs that select for a different spectrum of resistance mutations could add to the armamentarium that canbe used in the global effort to treat HIV-1 infections. It would be even better if some of the mutations these new compounds selected for would enhance the susceptibility of the virus to other drugs that target the same enzyme. ____Despite advances in HIV-1 treatment, drug resistance is still a problem. Of the four enzymatic activities found in HIV-1 proteins (protease, RT polymerase, RT RNase H, and IN), only RNase H has no approved therapeutics directed against it. This new target could be used to design and develop new classes of inhibitors that would suppress the replication of the drug-resistant variants that have been selected by the current therapeutics. We recently reported analysis of three compounds that selectively inhibit the RNase H of HIV-1 RT. The compounds also reduced the polymerase activity of RT; this ability was a result of the compounds binding to the RNase H active site. These compounds appear to be relatively specific; they do not inhibit either human RNase HI or human RNase H2. The compounds inhibit the replication of an HIV-1-based vector in a one-round assay, and their potencies were only modestly decreased by mutations that confer resistance to IN strand transfer inhibitors (INSTIs), NRTIs, or NNRTIs, suggesting that their ability to block HIV replication is related to their ability to block RNase H cleavage. These compounds appear to be useful leads that can be used to develop more potent and specific compounds. _____PATENTS LINKED TO THIS PROJECT: (1) Hughes S, Boyer P, Linial M, Stenbak C, Clark P: Foamy Virus Mutant Reverse Transcriptase. U.S. Patent #7,560,117, issued July 14, 2009. (2) Vu BC, Siddiqui MA, Marquez VE, Hughes SH, Boyer PL: C4'-Substituted-2-Deoxyadenosine Analogs and Methods of Treating HIV. U.S. Patent #8,513,214, issued August 20, 2013.